High Cell Density Cultivation of HEK293 Cells Using a 2L Membrane-Stirred Bioreactor.
The use of perfusion systems for the continuous cultivation of animal cells at high cell density (HCD) for the production of recombinant proteins, viruses, and viral vectors has many advantages. Besides an enhancement of volumetric productivity and high cell viability, the product quality can be improved through a steady-state environment. However, the establishment of such a process intensification method using stirred tank bioreactors (STRs) for cultivations at very high cell concentrations (> 50 × 106 cells mL-1) can be challenging. In particular, limitations in the oxygen supply, CO2 stripping, high viscosity, and excessive foam formation can impede cell viability and product yields. In this study, we investigated a novel membrane-stirrer (MemStir) based bioreactor that employs multiple hollow-fiber membrane sheets arranged in a device that facilitates efficient gas transfer through bubble-free diffusion for HCD cultivations. We found that the oxygen transfer rates at different tip speeds and gas flow rates resulted in volumetric mass transfer coefficient (kLa) values of 10-30 h-1 enabling sufficient oxygen supply to support concentrations exceeding 50 × 106 cells mL-1. The functionality of the membrane-stirrer was benchmarked in batch mode against shake flasks and a conventional STR equipped with a pitched-blade impeller and an l-drilled hole sparger. Suspension growth of a human embryonic kidney 293 LTV (HEK-LTV) cell in the MemStir system was comparable to the cell growth in a STR. Using pure oxygen for aeration, a maximum viable cell concentration of 7.6 × 106 cells mL-1 was achieved. As a starting point for further optimization, we investigated the impact of the MemStir system on viable cell concentration, cell viability, dissolved oxygen partial pressure, pH control and foam formation in HCD cultivations in perfusion mode. Using the MemStir system equipped with an alternating tangential flow system, HEK-LTV cells reached concentrations of up to 92 × 106 cells mL-1 with a specific growth rate of 0.022 h-1 in perfusion mode. Moreover, controlling the pH in the range 7.0 to 7.3 was easily achieved, and no foam formation was observed. Overall, these results suggest that the MemStir system is a viable option for HCD cultivation because it can overcome limitations of both aeration and foam formation.